Disorders of the cardiac conduction system can cause arrhythmias which is the major causes of sudden cardiac death in the patients with cardiovascular diseases. A deep understanding of CCS development and maintenance will likely offer new therapeutic strategies for prevention and therapy of arrhythmias. The PI previously described two muscle-specific miRNAs, miR-1-1 and miR-1-2, that are encoded by distinct genomic loci but share an identical mature sequence. The PI recently found that both miR- 1s are enriched in the CCS progenitors during cardiogenesis, and that both continue to be highly expressed in the whole CCS throughout development and in adulthood. miR-1s repress cell proliferation. Overexpression of miR-1-2 causes bradycardia and arrhythmias. Surviving miR-1-2 null mice exhibit bradycardia and develop cardiac conduction defects after birth but are without obvious cardiac structural abnormalities. The cardiac conduction defects in the miR-1-2 null adults are analogous to bundle branch block in patients, which are caused by abnormalities in the VCS. The PI hypothesizes that miR-1-2 plays a key role in restricting the cell cycle progression of CCS cells, and that it is a critical regulator controlling proper differentiation and maintenance of various CCS lineages. To test these hypotheses, the PI proposes to determine the role of miR-1-2 in regulating specification and proliferation of the CCS (Specific Aim 1); to determine how miR-1-2 establishes a precise level of calcium signaling, ion channels, and connexins during differentiation and maintenance of the CCS (Specific Aim 2); to identify affected genetic pathways upon loss of miR- 1-2 that are critical for development and maintenance of the CCS (Specific Aim 3). Completion of the proposed studies will elucidate new fundamental mechanism underlying development and function of the CCS, which will provide insights into cardiac arrhythmias and inform the development of novel strategies for treating them. PUBLIC HEALTH RELEVANCE: Understanding of the role of miRNA-mediated gene regulation may uncover new fundamental mechanisms of heart disease and help to develop strategies for treating heart disease.